Ornithine decarboxylase (ODC), the initial, rate-limiting enzyne in polyamine biosynthesis, has been established by somatic cell genetic and pharmacologic studies to be essential for cell proliferation We have demonstrated that ODC exists in murine RAW264 cells in phosphorylated and unphosphorylated isoforms and that both serine and threonine residues are phosphorylated. Cellular phosphorylation of ODC is regulated, and increased ODC phosphorylation is accompanied by an apparent increase in the enzyme's activity. Reduction of ODC phosphorylation in vitro results in a parallel loss of ODC phosphate and ODC activity. These results suggest that phosphorylated ODC is more active than unphosphorylated ODC. Furthermore, an additional regulatory consequence of ODC is suggested by the finding that in the intact cell, the unphosphorylated ODC is degraded at a faster rate than the phosphorylated ODC. The hypothesis to be tested in this proposal is that phosphorylation of ODC is an important mechanism for regulation of enzyme activity and/or the cellular level of enzyme protein via control of its degradation. The phosphorylated tryptic peptides from intracellularly phosphorylated ODC will be isolated and sequenced to identify the amino acids phosphorylated in situ. Site-directed mutagenesis will be used to create expression vectors for ODC with the intracellular phosphorylation sites modified and these will be transfected into a ODC-minus CHO cell line (putrescine auxotroph) to assess the effect of eliminating phosphorylation of ODC on cellular expression of ODC activity and on the process of intracellular degradation of the enzyme protein. The protein kinase(s) responsible for the intracellular phosphorylation of ODC will be identified, as well as the protein phosphatase(s), and the effects of phosphorylation and dephosphorylation of the purified ODC molecule will be assessed in vitro with respect to ODC activity. The possible interconversion of the ODC isoforms will be determined after loading of the ODC-minus CHO cell line with phospho- or unphospho-ODC by red blood cell-mediated cell fusion. These accomplishments will add to our understanding of the multiple mechanisms of intracellular control involved in regulation of ODC expression. In addition, they may suggest new pharmacologic approaches to inhibit essential ODC expressed in diseases such as parasitic and pneumocytis carinii infections and cancers.